Plants resistant to WT strains of cucumber mosaic virus

ABSTRACT

CP genes of CMV strains V27, V33, V34, and A35 (CMV-V27, CMV-V33, CMV-V34, and CMV-A35 respectively) are provided.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to coat protein (CP) genes derived from WTstrains of cucumber mosaic virus (CMV). More specifically, the inventionrelates to the genetic engineering of plants and to a method forconferring viral resistance to a plant using an expression cassetteencoding CP genes of WT strains of CMV.

[0003] 2. Description of the Prior Art

[0004] Many agriculturally important crops are susceptible to infectionby plant viruses, particularly CMV, which can seriously damage a crop,reduce its economic value to the grower, and increase its cost to theconsumer. Attempts to control or prevent infection of a crop by a plantvirus such as CMV have been made, yet viral pathogens continue to be asignificant problem in agriculture.

[0005] Scientists have recently developed means to produce virusresistant plants using genetic engineering techniques. Such an approachis advantageous in that the genetic material which provides theprotection is incorporated into the genome of the plant itself and canbe passed on to its progeny. A host plant is resistant if it possessesthe ability to suppress or retard the multiplication of a virus, or thedevelopment of pathogenic symptoms. “Resistant” is the opposite of“susceptible,” and may be divided into: (1) high, (2) moderate, or (3)low resistance, depending upon its effectiveness. Essentially, aresistant plant shows reduced or no symptom expression, and virusmultiplication within it is reduced or negligible. Several differenttypes of host resistance to viruses are recognized. The host may beresistant to: (1) establishment of infection, (2) virus multiplication,or (3) viral movement.

[0006] CMV is a single-stranded (+) ribonucleic acid (RNA) plant virusthat has a functionally divided genome. The virus genome contains fourRNA species designated RNAs 1-4. RNAs 3 and 4 encode the coat protein(CP) which is a protein that surrounds the viral RNA and protects theviral RNA from being degraded. Only RNAs 1-3 are required forinfectivity because the CP, which is encoded by RNA 4, is also encodedby RNA 3.

[0007] Several strains of CMV have been classified using serology, hostrange, peptide mapping, nucleic acid hybridization, and sequencinganalyses. These CMV strains can be divided into two groups, which aredesignated “WT” (also known as subgroup I) and “S” (also known assubgroup II). The S group consists of at least three members. The WTgroup is known to contain at least 17 members.

[0008] Expression of the CP genes from tobacco mosaic virus, alfalfamosaic virus, CMV, and potato virus X, among others, in transgenicplants has resulted in plants which are resistant to infection by therespective virus. Heterologous protection can also occur. For example,the expression of CP genes from watermelon mosaic virus-2 (WMV-2) orzucchini yellow mosaic virus (ZYMV) in transgenic tobacco plants hasbeen shown to confer protection against six other potyviruses: beanyellow mosaic virus, potato virus Y, pea mosaic virus, clover yellowvein virus, pepper mottle virus, and tobacco etch virus. However,expression of a preselected CP gene does not reliably conferheterologous protection to a plant. For example, transgenic squashplants containing the CMV-C CP gene, a WT virus, which have been shownto be resistant to the CMV-C strain are not protected to the same degreeagainst several other, highly virulent WT strains of CMV.

[0009] Thus, a need exists for plants resistant to WT strains of CMV.

SUMMARY OF THE INVENTION

[0010] This invention provides: an isolated and purifieddeoxyribonucleic acid (DNA) molecule that encodes the CP for the V27strain of CMV (CMV-V27), and a chimeric expression cassette comprisingthis DNA molecule; an isolated and purified DNA molecule that encodesthe CP for the V33 strain of CMV (CMV-V33), and a chimeric expressioncassette comprising this DNA molecule; and an isolated and purified DNAmolecule that encodes the CP for the V34 strain of CMV (CMV-V34), and achimeric expression cassette comprising this DNA molecule; and anisolated and purified DNA molecule that encodes the CP for the A35strain of CMV (CMV-A35), and a chimeric expression cassette comprisingthe DNA molecule. Another embodiment of the invention is exemplified bythe insertion of multiple virus gene expression cassettes into onepurified DNA molecule, e.g., a plasmid. Each of these cassettes alsoincludes a promoter which functions in plant cells to cause theproduction of an RNA molecule, and at least one polyadenylation signalcomprising 3′ nontranslated DNA which functions in plant cells to causethe termination of transcription and the addition of polyadenylatedribonucleotides to the 3′ end of the transcribed messenger RNA (mRNA)sequences, wherein the promoter is operably linked to the DNA molecule,and the DNA molecule is operably linked to the polyadenylation signal.Preferably, these cassettes include the promoter of the 35S gene ofcauliflower mosaic virus (CaMV-355 gene) and the polyadenylation signalof the CaMV-35S gene (CaMV-35S).

[0011] Also provided are bacterial cells, and transformed plant cells,containing the chimeric expression cassettes comprising the CP genesderived from the CMV-V27, CMV-V33, CMV-V34, or CMV-A35 strains, andpreferably the 35S promoter and the polyadenylation signal of theCaMV-35S gene. Plants are also provided, wherein the plants comprise aplurality of transformed cells containing the chimeric CP geneexpression cassettes derived from the CMV-V27, CMV-V33, CMV-V34, orCMV-A35 stains, and preferably the promoter and the polyadenylationsignal of the CaMV gene. Transformed plants of this invention includetobacco, beets, corn, cucumber, peppers, potatoes, melons, soybean,squash, and tomatoes. Especially preferred are members of theCucurbitaceae (e.g., squash and cucumber,) and Solanaceae (e.g., peppersand tomatoes) family.

[0012] Another aspect of the present invention is a method of preparinga CMV-resistant plant, such as a dicot, comprising: transforming plantcells with a chimeric expression cassette comprising a promoterfunctional in plant cells operably linked to a DNA molecule that encodesa CP of a WT strain of CMV, e.g., V27, V33, V34, or A35; regeneratingthe plant cells to provide a differentiated plant; and identifying atransformed plant that expresses the CMV CP at a level sufficient torender the plant resistant to infection by the specific strains of CMVdisclosed herein.

[0013] As used herein, with respect to a DNA molecule or “gene,” thephrase “isolated and purified” is defined to mean that the molecule iseither extracted from its context in the viral genome by chemical meansand purified and/or modified to the extent that it can be introducedinto the present vectors in the appropriate orientation, i.e., sense orantisense. As used herein, the term “chimeric” refers to the linkage oftwo or more DNA molecules which are derived from different sources,strains or species (e.g., from bacteria and plants), or the linkage oftwo or more DNA molecules, which are derived from the same species andwhich are linked in a way that does not occur in the native genome. Asused herein, “expression” is defined to mean transcription ortranscription followed by translation of a particular DNA molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1A. The nucleotide sequence of the CP gene of CMV-V27 fromnucleotide position 1 to 360. The deduced amino acid sequence of theencoded open reading frame is shown below the nucleotide sequence.

[0015]FIG. 1B. The nucleotide sequence of the CP gene of CMV-V27 fromnucleotide position 361 to 772. The deduced amino acid sequence of theencoded open reading frame is shown below the nucleotide sequence.

[0016]FIG. 2A. The nucleotide sequence of the CP gene of CMV-V33 fromnucleotide position 1 to 420. The deduced amino acid sequence of theencoded open reading frame is shown below the nucleotide sequence.

[0017]FIG. 2B. The nucleotide sequence of the CP gene of CMV-V33 fromnucleotide position 421 to 773. The deduced amino acid sequence of theencoded open reading frame is shown below the nucleotide sequence.

[0018]FIG. 3. The nucleotide sequence of the CP gene of CMV-V34 fromnucleotide position 1 to 771. The deduced amino acid sequence of theencoded open reading frame is shown below the nucleotide sequence.

[0019]FIG. 4A. The alignment of the nucleotide sequences of the CP genesfrom five CMV strains from nucleotide position 1 to 600. Ccp and Cmvwlare described in Quemada et al. (J. Gen. Virol., 70:1065 (1989)).Alignments were obtained with the use of the UWGCG Pileup program. Thedots represent either the lack of sequence information at the 5′ end ofthe CP gene or gaps in homology in sequences relative to others in thealignment. The position of primer RMM351 is shown.

[0020]FIG. 4B. The alignment of the nucleotide sequences of the CP genesfrom five CMV strains described in FIG. 4A from nucleotide position 601to 840.

[0021]FIG. 4C. The alignment of the nucleotide sequences of the CP genesfrom five CMV strains described in FIG. 4A from nucleotide position 841to 1080.

[0022]FIG. 4D. The alignment of the nucleotide sequences of the CP genesfrom five CMV strains from nucleotide position 1081 to 1170 alignment.The position of primer RMM352 is shown.

[0023]FIG. 5A. The alignment of the sequences of amino acid 1-150deduced from the nucleotide sequences of CMV strains V27, V33, V34,CMV-C (shown in FIG. 4) and CMV strain Cmvq3 (Quemada et al., J. Gen.Virol., 70:1065 (1989)). Alignments were performed by the UWGCG Pileupprogram. Differences among the WT virus strains are underlined andhighlighted with asterisks. The dots represent gaps in homology insequences relative to others in the alignment.

[0024]FIG. 5B. The alignment of the sequences of amino acid 151-219deduced from the nucleotide sequences of CMV strains as described inFIG. 5A.

[0025]FIG. 6A. Assembly of CMV-V27 CP expression cassette. Polymerasechain reaction (PCR) products of CMV-V27 were installed into pCRII andsubsequently inserted into pUC18cpexpress by routine methods. The boldedlines and arrows which are a part of the circle represent CaMV-35Ssequences.

[0026]FIG. 6B. (FIG. 6A, continued.) Insertion of a CMV-V27 CPexpression cassette BamHI fragment into the BglII site of pEPG204 andpEPG205 to produce pEPG239 and pEPG240, respectively.

[0027]FIG. 6C. Restriction map of pEPG239. This binary plasmid includesthe CP expression cassettes for PRV (melon, long), CMV-V27, ZYMV, andWMV-2. For further information on PRV CP genes, refer to Applicants'International Patent Application No. PCT/US95/07272 entitled “PapayaRingspot Virus Coat Protein Gene” filed on Jun. 7, 1995, incorporated byreference herein. For further information on ZYMV and WMV-2 CP genes,refer to Applicants' International Patent Application No. PCT/US89/03094filed on Jul. 20, 1989 entitled “Potyvirus Coat Protein Genes and PlantsTransformed Therewith”, incorporated by reference herein.

[0028]FIG. 6D. Restriction map of pEPG240. This binary plasmid includesthe CP expression cassettes for PRV (melon, short), CMV-V27, ZYMV, andWMV-2.

[0029]FIG. 7A. Assembly of CMV-V33 CP expression cassette. PCR productsof CMV-V33 were installed into pUC1318cpexpress by routine methods.

[0030]FIG. 7B. (FIG. 7A, continued.) Insertion of a CMV-V33 CPexpression cassette BamHI fragment into the BglII site of pEPG204 andpEPG205 to produce pEPG196 and pEPG197, respectively.

[0031]FIG. 7C. Restriction map of pEPG196. This binary plasmid includesthe CP expression cassettes for PRV (melon, long), CMV-V33, ZYMV, andWMV-2. Arrows indicate CaMV-35S promoter fragments.

[0032]FIG. 7D. Restriction map of pEPG197. This binary plasmid includesthe CP expression cassettes for PRV (melon, short), CMV-V33, ZYMV, andWMV-2.

[0033]FIG. 8. The nucleotide sequence of the CP gene of CMV-A35. Thededuced amino acid sequence of the encoded open reading frame is shownbelow the nucleotide sequence.

[0034]FIG. 9A. The alignment of the amino acid sequences deduced fromthe nucleotide sequences of the six CMV strains shown in FIG. 10A foramino acid 1-120. Differences among the “C” type viruses are enclosed inboxes. The dashes represent gaps in homology in sequences relative toothers in the alignment.

[0035]FIG. 9B. The alignment of the amino acid sequences deduced fromthe nucleotide sequences of the six CMV strains shown in FIG. 10 foramino acid 121 to 220.

[0036]FIG. 10A. The alignment of the nucleotide sequences of the CPgenes from 6 CMV strains from nucleotide position 321-400 of a consensussequence. The dots represent either the lack of sequence information atthe 5′ end of the CP gene or gaps in homology in sequences relative toothers in the alignment.

[0037]FIG. 10B. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 401to 480.

[0038]FIG. 10C. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 481to 560.

[0039]FIG. 10D. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 561to 640.

[0040]FIG. 10E. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 641to 720.

[0041]FIG. 10F. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 721to 800.

[0042]FIG. 10G. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 801to 880.

[0043]FIG. 10H. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 881to 960.

[0044]FIG. 10I. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 961to 1040.

[0045]FIG. 10J. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 1041to 1120.

[0046]FIG. 10K. The alignment of the nucleotide sequences of the CPgenes of CMV strains described in FIG. 10A from nucleotide position 1121to 1200. The dots represent gaps in homology in sequences relative toothers in the alignment.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The genome of CMV contains four RNA species designated RNA 1, 2,3 and 4; 3389 nucleotides (nt), 3035 nt, 2193 nt, and 1027 nt,respectively (Peden et al., Virol., 53:487 (1973); Gould et al., Eur. J.Biochem., 126:217 (1982); Rezaian et al., Eur. J. Biochem., 143:227(1984); Rezaian et al., Eur. J. Biochem. 150:331 (1985)). Only RNA 1, 2and 3 are required for infectivity (Peden et al., Virol., 53:487 (1973))because the CP, which is encoded by RNA 4, is also encoded by RNA 3.Translation of CMV RNA yield a 95 kiloDalton (kD) polypeptide from RNA1, a 94 kD polypeptide from RNA 2 (Gordon et al., Virol., 123:284(1983)), and two polypeptides from RNA 3: its 5′ end encodes a 35 kDpolypeptide, and its 3′ end encodes a 24.5 kD polypeptide (Gould et al.,Eur. J. Biochem., 126:217 (1982)). The 24.5 kD polypeptide is identicalto that encoded by RNA 4 and is the CP.

[0048] Several strains of CMV have been classified using serology, hostrange, peptide mapping, nucleic acid hybridization, and sequencing.These CMV strains include two groups, WT and S. CMV WT strains includeCMV-C, CMV-V27, CMV-V33, CMV-V34, CMV-M, CMV-O, CMV-Y, and CMV-A35 whileS strains include CMV-Q, CMV-WL, and CMV-LS (Zaitlin et al., Virol.,201:200 (1994)). Protection against a strain in one group does notnecessarily provide protection against all strains in that group. Forexample, transgenic squash plants protected with CP genes from the CMV-Care not protected against the CMV strains V27, V33, V34, or A35. Inaddition, Zaitlin et al. (Virol., 201:200 (1994)) report that tobaccoplants transgenic for a CMV-FNY replicase gene show protection againstchallenge from WT strains but show no protection against challenge fromS strain challenges. Thus, the present invention is directed toproviding plants with resistance to WT strains of CMV, e.g., V27, V33,V34, or A35.

[0049] To practice the present invention, a viral gene must be isolatedfrom the viral genome and inserted into a vector. Thus, the presentinvention provides isolated and purified DNA molecules that encode theCP of the V27, V33, or V34 strains of CMV. As used herein, a DNAmolecule that encodes a CP gene includes nucleotides of the codingstrand, also referred to as the “sense” strand, as well as nucleotidesof the noncoding strand, complementary strand, also referred to as the“antisense” strand, either alone or in their base-paired configuration.Thus, a DNA molecule that encodes the CP of the V27 strain of CMV, forexample, includes the DNA molecule having the nucleotide sequence ofFIG. 1, a DNA molecule complementary to the nucleotide sequence of FIG.1, as well as a DNA molecule which also encodes a CMV CP and itscomplement which hybridizes with a CMV-V27-specific DNA probe inhybridization buffer with 6XSSC, 5X Denhardt's reagent, 0.5% SDS and 100micrograms per milliliter (μg/ml) denatured, fragmented salmon sperm DNAand remains bound when washed at 68° C. in 0.1XSSC and 0.5% SDS(Sambrook et al., Molecular Clonging: A Laboratory Manual, 2nd ed.(1989)). Moreover, the DNA molecules of the present invention caninclude non-CMV CP nucleotides that do not interfere with expression ofthe CMV CP gene. Preferably, the isolated and purified DNA molecules ofthe present invention comprise a single coding region for the CP. Thus,preferably the DNA molecules of the present invention are thoseconsisting essentially of DNA that encodes the CP.

[0050] These CMV genes are used to produce the CPs, which are believedto confer resistance to viruses. Another molecular strategy to providevirus resistance in transgenic plants is based on antisense RNA. As iswell known, a cell manufactures protein by transcribing the DNA of thegene encoding that protein to produce RNA, which is then processed tomRNA (e.g., by the removal of introns) and finally translated byribosomes into protein. This process may be inhibited in the cell by thepresence of antisense RNA. The term antisense RNA means an RNA sequencewhich is complementary to a sequence of bases in the mRNA in question inthe sense that each base (or the majority of bases) in the antisensesequence (read in the 3′ to 5′ sense) is capable of pairing with thecorresponding base (G with C, A with U) in the mRNA sequence read in the5′ to 3′ sense. It is believed that this inhibition takes place byformation of a complex between the two complementary strands of RNA,thus preventing the formation of protein. How this works is uncertain:the complex may interfere with further transcription, processing,transport or translation, or degrade the mRNA, or have more than one ofthese effects. This antisense RNA may be produced in the cell bytransformation of the cell with an appropriate DNA construct arranged totranscribe the non-template strand (as opposed to the template strand)of the relevant gene (or of a DNA sequence showing substantial homologytherewith).

[0051] The use of antisense RNA to downregulate the expression ofspecific plant genes is well known. Reduction of gene expression has ledto a change in the phenotype of the plant: either at the level of grossvisible phenotypic difference, e.g., lack of anthocyanin production inflower petals of petunia leading to colorless instead of colored petals(van der Krol et al., Nature, 333:866-869 (1988)); or at a more subtlebiochemical level, e.g., change in the amount of polygalacturonase andreduction in depolymerization of pectin during tomato fruit ripening(Smith et al., Nature, 334:724-726 (1988)).

[0052] Another more recently described method of inhibiting geneexpression in transgenic plants is the use of sense RNA transcribed froman exogenous template to downregulate the expression of specific plantgenes (Jorgensen, Keystone Symposium “Improved Crop and Plant Productsthrough Biotechnology”, Abstract X1-022 (1994)). Thus, both antisenseand sense RNA have been proven to be useful in achieving downregulationof gene expression in plants, which are encompassed by the presentinvention.

[0053] The CMV CP gene does not contain the signals necessary for itsexpression once transferred and integrated into a plant genome.Accordingly, a vector must be constructed to provide the regulatorysequences such that they will be functional upon inserting a desiredgene. When the expression vector/insert construct is assembled, it isused to transform plant cells which are then used to regenerate plants.These transgenic plants carry the viral gene in the expressionvector/insert construct. The gene is expressed in the plant andincreased resistance to viral infection is conferred thereby.

[0054] Several different methods exist to isolate a viral gene. To doso, one having ordinary skill in the art can use information about thegenomic organization of cucumoviruses to locate and isolate the CP gene.The CP gene is located near the 3′ end of RNA 3. Using methods wellknown in the art, a quantity of virus is grown and harvested. The viralRNA is then separated by gel electrophoresis. A cDNA library is createdusing the viral RNA, by methods known to the art. The viral RNA isincubated with primers that hybridize to the viral RNA and reversetranscriptase, and a complementary DNA molecule is produced. A DNAcomplement of the complementary DNA molecule is produced and thatsequence represents a DNA copy (cDNA) of the original viral RNAmolecule. The DNA complement can be produced in a manner that results ina single double stranded cDNA or PCR can be used to amplify the DNAencoding the cDNA with the use of oligomer primers specific for viralsequences. These primers can include novel restriction sites used insubsequent cloning steps. Thus, a double stranded DNA molecule isgenerated which contains the sequence information of the viral RNA.These DNA molecules can be cloned in E. coli plasmid vectors after theadditions of restriction enzyme linker molecules by DNA ligase. Thevarious fragments are inserted into cloning vectors, such aswell-characterized plasmids, which are then used to transform E. coliand create a cDNA library. CMV CP genes from previously isolated strainscan be used as hybridization probes to screen the cDNA library todetermine if any of the transformed bacteria contain DNA fragments withsequences coding for a CMV CP. Alternatively, plasmids which harbor CMVCP sequences can be determined by restriction enzyme digestion ofplasmids in bacterial transformants. The cDNA inserts in any bacterialcolonies which contain this region can be sequenced. The CP gene ispresent in its entirety in colonies which have sequences that extend 5′to the sequence which encodes the ATG start codon and sequences thatextend 3′ of the stop codon.

[0055] Alternatively, cDNA fragments can be inserted in the senseorientation into expression vectors. Antibodies against the CP can beused to screen the cDNA expression library and the gene can be isolatedfrom colonies which express the protein.

[0056] In the present invention, the DNA molecules encoding the CP genesof the CMV strains V27, V33, V34, and A35 have been determined and thegenes have been inserted into expression cassettes. These expressioncassettes can be individually placed into a vector that can betransmitted into plants, preferably a binary vector. Alternatively, twoor more of the CMV CP genes can each be present in an expressioncassette which can be placed into the same binary vector, or any of theCMV CP expression cassettes of the present invention can be placed intoa binary vector with one or more viral gene expression cassettes. Theexpression vectors contain the necessary genetic regulatory sequencesfor expression of an inserted gene. The CP gene is inserted such thatthose regulatory sequences are functional and the genes can be expressedwhen incorporated into a plant genome. For example, vectors of thepresent invention can contain combinations of expression cassettes thatinclude DNA from a heterologous CMV CP gene (i.e., one from another CMVisolate), papaya ringspot virus (PRV) CP gene, a ZYMV CP gene, and aWMV-2 CP gene.

[0057] Moreover, when combinations of viral gene expression cassettesare placed in the same binary plasmid, and that multigene cassettecontaining plasmid transformed into a plant, the viral genes allpreferably exhibit substantially the same degrees of efficacy whenpresent in transgenic plants. For example, if one examines numeroustransgenic lines containing two different intact viral gene cassettes,the transgenic line will be immune to infection by both viruses.Similarly, if a line exhibits a delay in symptom development to onevirus, it will also exhibit a delay in symptom development to the secondvirus. Finally, if a line is susceptible to one of the viruses it willbe susceptible to the other. This phenomenon is unexpected. If therewere not a correlation between the efficacy of each gene in thesemultiple gene constructs this approach as a tool in plant breeding wouldprobably be prohibitively difficult to use. Even with single geneconstructs, one must test numerous transgenic plant lines to find onethat displays the appropriate level of efficacy. The probability offinding a line with useful levels of expression can range from 10-50%(depending on the species involved). For further information refer toApplicants' International Patent Application No. PCT/US95/06261 entitled“Transgenic Plants Expressing DNA Constructs Containing a Plurality ofGenes to Impart Virus Resistance” filed on Jun. 7, 1995, incorporated byreference herein.

[0058] In order to express the viral gene, the necessary geneticregulatory sequences must be provided. In the present invention, the CPgenes are inserted into vectors which contain cloning sites forinsertion 3′ of the initiation codon and 5′ of the poly(A) signal. Thepromoter is 5′ of the initiation codon such that when genes are insertedat the cloning site, a functional unit is formed in which the insertedgenes are expressed under the control of the various genetic regulatorysequences.

[0059] The segment of DNA referred to as the promoter is responsible forthe regulation of the transcription of DNA into mRNA. A number ofpromoters which function in plant cells are known in the art and can beemployed in the practice of the present invention. These promoters canbe obtained from a variety of sources such as plants or plant viruses,and can include, but are not limited to, promoters isolated from thecaulimovirus group such as the CaMV-35S promoter (CaMV-35S), theenhanced CaMV-35S promoter (enh-CaMV-35S), the figwort mosaic virusfull-length transcript promoter (FMV-35S), and the promoter isolatedfrom the chlorophyll a/b binding protein. Other useful promoters includepromoters which are capable of expressing the cucumovirus proteins in aninducible manner or in a tissue-specific manner in certain cell types inwhich the infection is known to occur. For example, the induciblepromoters from phenylalanine ammonia lyase, chalcone synthase,hydroxyproline rich glycoprotein, extensin, pathogenesis-relatedproteins (e.g. PR-1a), and wound-inducible protease inhibitor frompotato may be useful.

[0060] Preferred promoters for use in the present CP-containingcassettes include the constitutive promoters from CaMV, thetumor-inducing (Ti) genes nopaline synthase (NOS) (Bevan et al., NucleicAcids Res., 11:369 (1983)) and octopine synthase (Depicker et al., J.Mol. Appl. Genet., 1:561 (1982)), and the bean storage protein genephaseolin. The poly(A) addition signals from these genes are alsosuitable for use in the present cassettes. The particular promoterselected is preferably capable of causing sufficient expression of theDNA coding sequences to which it is operably linked, to result in theproduction of amounts of the proteins or RNA effective to provide viralresistance, but not so much as to be detrimental to the cell in whichthey are expressed. The promoters selected should be capable offunctioning in tissues including, but not limited to, epidermal,vascular, and mesophyll tissues. The actual choice of the promoter isnot critical, as long as it has sufficient transcriptional activity toaccomplish the expression of the preselected proteins or theirrespective RNAs and subsequent conferral of viral resistance to theplants.

[0061] The nontranslated leader sequence can be derived from anysuitable source and can be specifically modified to increase thetranslation of the mRNA. The 5′ nontranslated region can be obtainedfrom the promoter selected to express the gene, an unrelated promoter,the native leader sequence of the gene or coding region to be expressed,viral RNAs, suitable eucaryotic genes, or a synthetic gene sequence. Thepresent invention is not limited to the constructs presented in thefollowing examples.

[0062] The termination region or 3′ nontranslated region which isemployed is one which will cause the termination of transcription andthe addition of polyadenylated ribonucleotides to the 3′ end of thetranscribed mRNA sequence. The termination region can be native with thepromoter region, native with the gene, or can be derived from anothersource, and preferably include a terminator and a sequence coding forpolyadenylation. Suitable 3′ nontranslated regions of the chimeric plantgene include but are not limited to: (1) the 3′ transcribed,nontranslated regions containing the polyadenylation signal ofAgrobacterium Ti plasmid genes, such as the NOS gene; and (2) plantgenes like the soybean 7S storage protein genes.

[0063] Preferably, the expression cassettes of the present invention areengineered to contain a constitutive promoter 5′ to its translationinitiation codon (ATG) and a poly(A) addition signal (AATAAA) 3′ to itstranslation termination codon. Several promoters which function inplants are available, however, the preferred promoter is the 35Sconstitutive promoters from CaMV. The poly (A) signal can be obtainedfrom the CaMV-35S gene or from any number of well characterized plantgenes, i.e., NOS, octopine synthase, and the bean storage protein genephaseolin. The constructions are similar to that used for the expressionof the CMV-C CP in PCT Patent Application PCT/US88/04321, published onJun. 29, 1989 as WO 89/05858, claiming the benefit of U.S. SN 135,591,filed De. 21, 1987, entitled “Cucumber Mosaic Virus Coat Protein Gene”,and the CMV WL CP in PCT Patent Application PCT/US89/03288, published onMar. 8, 1990 as WO 90/02185, claiming the benefit of U.S. SN 234,404,filed Au. 19, 1988, entitled “Cucumber Mosaic Virus Coat Protein Gene.”

[0064] Selectable marker genes can be incorporated into the presentexpression cassettes and used to select for those cells or plants whichhave become transformed. The marker gene employed may express resistanceto an antibiotic, such as kanamycin, gentamycin, G418, hygromycin,streptomycin, spectinomycin, tetracycline, chloramphenicol, and thelike. Other markers could be employed in addition to or in thealternative, such as, for example, a gene coding for herbicide tolerancesuch as tolerance to glyphosate, sulfonylurea, phosphinothricin, orbromoxynil. Additional means of selection could include resistance tomethotrexate, heavy metals, complementation providing prototrophy to anauxotrophic host, and the like.

[0065] The particular marker employed will be one which will allow forthe selection of transformed cells as opposed to those cells which arenot transformed. Depending on the number of different host species oneor more markers can be employed, where different conditions of selectionwould be useful to select the different host, and would be known tothose of skill in the art. A screenable marker such as theβ-glucuronidase gene can be used in place of, or with, a selectablemarker. Cells transformed with this gene can be identified by theproduction of a blue product on treatment with5-bromo-4-chloro-3-indoyl-β-D-glucuronide.

[0066] In developing the present expression construct, i.e., expressioncassette, the various components of the expression construct such as theDNA molecules, linkers, or fragments thereof will normally be insertedinto a convenient cloning vector, such as a plasmid or phage, which iscapable of replication in a bacterial host, such as E. coli. Numerouscloning vectors exist that have been described in the literature. Aftereach cloning, the cloning vector can be isolated and subjected tofurther manipulation, such as restriction, insertion of new fragments,ligation, deletion, resection, insertion, in vitro mutagenesis, additionof polylinker fragments, and the like, in order to provide a vectorwhich will meet a particular need.

[0067] For Agrobacterium-mediated transformation, the expressioncassette will be included in a vector, and flanked by fragments of theAgrobacterium Ti or root-inducing (Ri) plasmid, representing the rightand, optionally the left, borders of the Ti or Ri plasmid transferredDNA (T-DNA). This facilitates integration of the present chimeric DNAsequences into the genome of the host plant cell. This vector will alsocontain sequences that facilitate replication of the plasmid inAgrobacterium cells, as well as in E. coli cells.

[0068] All DNA manipulations are typically carried out in E. coli cells,and the final plasmid bearing the cucumovirus expression cassette ismoved into Agrobacterium cells by direct DNA transformation,conjugation, and the like. These Agrobacterium cells will contain asecond plasmid, also derived from Ti or Ri plasmids. This second plasmidwill carry all the vir genes required for transfer of the foreign DNAinto plant cells. Suitable plant transformation cloning vectors includethose derived from a Ti plasmid of Agrobacterium tumefaciens, asgenerally disclosed in Glassman et al. (U.S. Pat. No. 5,258,300), orAgrobacterium rhizogenes.

[0069] A variety of techniques are available for the introduction of thegenetic material into or transformation of the plant cell host. However,the particular manner of introduction of the plant vector into the hostis not critical to the practice of the present invention, and any methodwhich provides for efficient transformation can be employed. In additionto transformation using plant transformation vectors derived from the Tior Ri plasmids of Agrobacterium, alternative methods could be used toinsert the DNA constructs of the present invention into plant cells.Such methods may include, for example, the use of liposomes,electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA, 82:824(1984)), chemicals that increase the free uptake of DNA (Paszkowski etal., EMBO J., 3:2717 (1984)), DNA delivery via microprojectilebombardment (Klein et al., Nature, 327:70 (1987)), microinjection(Crossway et al., Mol. Gen. Genet., 202:179 (1985)), and transformationusing viruses or pollen.

[0070] The choice of plant tissue source or cultured plant cells fortransformation will depend on the nature of the host plant and thetransformation protocol. Useful tissue sources include callus,suspension culture cells, protoplasts, leaf segments, stem segments,tassels, pollen, embryos, hypocotyls, tuber segments, meristematicregions, and the like. The tissue source is regenerable, in that it willretain the ability to regenerate whole, fertile plants followingtransformation.

[0071] The transformation is carried out under conditions directed tothe plant tissue of choice. The plant cells or tissue are exposed to theDNA carrying the present viral gene expression cassette(s) for aneffective period of time. This can range from a less-than-one-secondpulse of electricity for electroporation, to a two-to-three dayco-cultivation in the presence of plasmid-bearing Agrobacterium cells.Buffers and media used will also vary with the plant tissue source andtransformation protocol. Many transformation protocols employ a feederlayer of suspended culture cells (tobacco or Black Mexican Sweet Corn,for example) on the surface of solid media plates, separated by asterile filter paper disk from the plant cells or tissues beingtransformed.

[0072] Following treatment with DNA, the plant cells or tissue may becultivated for varying lengths of time prior to selection, or may beimmediately exposed to a selective agent such as those describedhereinabove. Protocols involving exposure to Agrobacterium will alsoinclude an agent inhibitory to the growth of the Agrobacterium cells.Commonly used compounds are antibiotics such as cefotaxime andcarbenicillin. The media used in the selection may be formulated tomaintain transformed callus or suspension culture cells in anundifferentiated state, or to allow production of shoots from callus,leaf or stem segments, tuber disks, and the like.

[0073] Cells or callus observed to be growing in the presence ofnormally inhibitory concentrations of the selective agents are presumedto be transformed and may be subcultured several additional times on thesame medium to remove nonresistant sections. The cells or calli can thenbe assayed for the presence of the viral gene cassette, or can besubjected to known plant regeneration protocols. In protocols involvingthe direct production of shoots, those shoots appearing on the selectivemedia are presumed to be transformed and can be excised and rooted,either on selective medium suitable for the production of roots, or bysimply dipping the excised shoot in an Ri compound and directly plantingit in vermiculite.

[0074] In order to produce transgenic plants exhibiting viralresistance, the viral genes must be taken up into the plant cell andstably integrated within the plant genome. Plant cells and tissuesselected for their resistance to an inhibitory agent are presumed tohave acquired the selectable marker gene encoding this resistance duringthe transformation treatment. Since the marker gene is commonly linkedto the viral genes, it can be assumed that the viral genes havesimilarly been acquired. Southern blot hybridization analysis using aprobe specific to the viral genes can then be used to confirm that theforeign genes have been taken up and integrated into the genome of theplant cell. This technique may also give some indication of the numberof copies of the gene that have been incorporated. Successfultranscription of the foreign gene into mRNA can likewise be assayedusing Northern blot hybridization analysis of total cellular RNA and/orcellular RNA that has been enriched in a polyadenylated region. mRNAmolecules encompassed within the scope of the invention are those whichcontain viral specific sequences derived from the viral genes present inthe transformed vector which are of the same polarity as that of theviral genomic RNA such that they are capable of base pairing with viralspecific RNA of the opposite polarity to that of viral genomic RNA underconditions described in Chapter 7 of Sambrook et al. (1989). Moreover,mRNA molecules encompassed within the scope of the invention are thosewhich contain viral specific sequences derived from the viral genespresent in the transformed vector which are of the opposite polarity asthat of the viral genomic RNA such that they are capable of base pairingwith viral genomic RNA under conditions described in Chapter 7 inSambrook et al. (1989).

[0075] The presence of a viral gene can also be detected byimmunological assays, such as the double-antibody sandwich assaysdescribed by Namba et al., Gene, 107:181 (1991) as modified by Clark etal., J. Gen. Virol., 34:475 (1979). See also, Namba et al.,Phytopathology, 82:940 (1992). Cucumovirus resistance can also beassayed via infectivity studies as generally disclosed by Namba et al.,ibid., wherein plants are scored as symptomatic when any inoculated leafshows vein clearing, mosaic or necrotic symptoms.

[0076] Seed from plants regenerated from tissue culture is grown in thefield and self-pollinated to generate true breeding plants. The progenyfrom these plants become true breeding lines which are evaluated forviral resistance in the field under a range of environmental conditions.The commercial value of viral-resistant plants is greatest if manydifferent hybrid combinations with resistance are available for sale.The farmer typically grows more than one kind of hybrid based on suchdifferences as maturity, color or other agronomic traits. Additionally,hybrids adapted to one part of a country are not adapted to another partbecause of differences in such traits as maturity, disease and insecttolerance. Because of this, it is necessary to breed viral resistanceinto a large number of parental lines so that many hybrid combinationscan be produced.

[0077] The invention will be further described by reference to thefollowing detailed examples. Enzymes were obtained from commercialsources and were used according to the vendor's recommendations or othervariations known in the art. Other reagents, buffers, etc., wereobtained from commercial sources, such as Sigma Chemical Co., St. Louis,Mo., unless otherwise specified.

[0078] Most of the recombinant DNA methods employed in practicing thepresent invention are standard procedures, well known to those skilledin the art, and described in detail in, for example, in European PatentApplication Publication Number 223,452, published Nov. 29, 1986, whichis incorporated herein by reference. General references containing suchstandard techniques include the following: R. Wu, ed., Methods inEnzymology, Vol. 68 (1979); J. H. Miller, Experiments in MolecularGenenetics (1972) ; J. Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed. (1989); and D. M. Glover, ed., DNA Cloning Vol. II(1982).

[0079]FIGS. 6 and 7 are presented to illustrate the constructions ofthis invention.

EXAMPLE 1

[0080] A. Isolation of CMV RNAs

[0081] Zucchini squash plants (20-day old) were inoculated with CMVstrains V27, V33, or V34; after 7-10 days, infected leaves wereharvested and CMV virus particles were isolated. The procedure used wasbased on protocols from Lot et al., Annals of Phytopathology, 4:25(1972), Francki et al., CMI/AAB Descriptions of Plant Viruses, (July,1979), and Habili and Francki, Virology, 57:292 (1974). Approximately100 grams (g) of fresh leaves were extracted in an equal weight pervolume (w/v) of 0.5 molar (M) Na-citrate (pH 6.5) containing 5millimolars (mM) EDTA and 100 milliliters (ml) of chloroform. Aftercentrifugation of the extract at 12,000×G for 10 minutes,polyethyleneglycol (“PEG”, Sigma Chemical Co. PEG-8000, averagemolecular weight, Research Grade) was added to the supernatant to afinal concentration of 10% and the suspension was stirred for 30-40minutes at 0-4° C. This suspension was centrifuged at 12,000×G for 10minutes, and the pellet was resuspended in 40-50 ml of 5 mM Na-boratebuffer (pH 9.0) containing 0.5 M EDTA. TRITON X-100 was then added tothe virus particle suspension to a final concentration of 2% and stirredon ice for 30 minutes. This suspension was then centrifuged at 19,000×Gfor 15 minutes, and the supernatant was collected and subsequentlycentrifuged at 105,000×G for 2 hours. The virus pellet was collected andresuspended in about 2 ml of 5 mM Na-borate buffer (pH 9.0) containing0.5 mM EDTA. The resuspended virus preparation was applied onto a stepsucrose gradient consisting of 5 layers: 5%, 10%, 15%, 20%, and 25%sucrose dissolved in 2.0 mM Na-phosphate buffer (pH 7.5). Gradients werecentrifuged at 37,000 rpm in a Sorvall TH641 swinging bucket rotor for45 minutes. After centrifugation, the virus band was harvested, thevirus preparation was dialyzed against Na-borate buffer, and LiCl wasadded (2 M final concentration) to lyse the virions and to precipitateviral RNA. CMV RNA was dissolved and reprecipitated with ethanol anddissolved in water. By agarose gel electrophoresis, the expected fourRNA species were observed.

[0082] B. Cloning CMV Coat Protein Genes

[0083] (a) CMV-V27

[0084] The first cDNA strand of CMV-V27 was synthesized with the use ofPerkin-Elmer RT-PCR kit reagents and the primer RMM352 (shown in FIG.4); immediately in the same reaction tube, a PCR was carried out withthe use of oligonucleotide primers RMM351 and RMM352 (shown in FIG. 4),following the manufacturer's protocol. The ATG translation start isincluded in the NcoI site present in primer RMM351. Individual PCRproduct molecules were cloned using the TA Cloning™ kit (InvitrogenCorp., San Diego, Calif.) into pCRII (included in the TA Cloning™ kit asa linearized plasmid with single 3′ dT overhangs at the ends of themolecule). Three clones were isolated for further study: CMVV27TA21,CMVV27TA23, and CMVV27TA26. With the use of a kit (Sequenase 2 purchasedfrom USB, Cleveland, Ohio), the CMV-V27 insert in clone CMVV27TA21 wassequenced.

[0085] CMV-V27 was compared to 11 different CMV isolates: Cmvbaul,Cmvq3, Cmvw1, Cmvtrk7, Cmvfc, Cmvi17f, Cmvc, Cmvpr50, Cmvv27, Cmvp6,Cmvo, Cmvm, and Cmvy. CMV-V27 CP is similar to CMV-Y in that it containsa serine at position 29 while other strains have an alanine at thisposition. However, CMV-Y contains a leucine at position 18 while CMV-V27contains a proline at position 18. In addition, CMV-V27 has a methionineat position 206, no other CMV-C group viruses have a methionine at thisposition (Baulcombe, D., “Mutational analysis of CMV RNA3: Effects onRNA3 accumulation, RNA4 synthesis and plant infection.” UnpublishedDirect Submission. Submitted (19-JUN-1992) David Baulcombe, TheSainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich, NR27UH, United Kingdom; Hayakawa et al., Gene, 71:107 (1988); Hayakawa etal., J. Gen. Virol. 70:499 (1989); Owen et al., J. Gen. Virol., 71:2243(1990); Pappu et al., “The nucleotide and the deduced amino acidsequences of CP genes of three Puerto Rican isolates of CMV.”Unpuglished (1992). This sequence is included in the GeneBank sequencedata base; Salanki et al., “Complete nucleotide sequence of RNA 3 fromCMV strain Trk 7.” Unpublished (1993) This sequence is included in theGeneBank data base; Shintaku, J. Gen. Virol. 72:2587 (1991)).

[0086] (b) CMV- V33

[0087] CMV-V33 was purified and viral RNA extracted from a virionpreparation as described above; subsequently single stranded cDNA wassynthesized using Perkin-Elmer RT-PCR kit reagents and oligomer primerRMM352. The CP gene of strain V33 was amplified using PCR as describedabove for V27 with the use of oligomer primers RMM351 and RMM352 (FIG.4). The V33 CP gene PCR product was digested with NcoI and directlycloned into the expression cassette cpexpress installed into pUC1318(see Kay and McPherson, Nucleic Acids Research, 15:2779 (1987) forpUC1318; Slightom, Gene, 100:251 (1991) for cpexpress; pUC1318cpexpressis the cpexpress described in Slightom, however it is installed into theHindIII site of the modified pUC plasmid pUC1318 described in detail inKay and McPherson), rather than into the intermediate vector pCRII. Bycolony hybridization with a CMV CP probe, a number of clones wereidentified for further analysis: V33cel, V33ce2, V33ce7, and V33ce9. TheCMV-V33 insert in clone V33ce7 was sequenced with the use of a kit(Sequenase 2 purchased from USB, Cleveland, Ohio).

[0088] CMV-V33 was compared to 11 different CMV isolates: Cmvbaul,Cmvq3, Cmvw1, Cmvtrk7, Cmvfc, Cmvi17f, Cmvc, Cmvpr50, Cmvv27, Cmvp6,Cmvo, Cmvm, and Cmvy. CMV-V33 has a serine at position 67 while allother CMV strains compared included a proline at this position. Atposition 196, both CMV-V33 and CMV-Y have a valine residue; all othermembers of the CMV-C group contains isoleucine at this position.However, at position 184, CMV-V33 has an alanine residue while CMV-Y hasa threonine residue. Therefore, CMV-V33 CP is unique (Baulcombe, D.,“Mutational analysis of CMV RNA3: Effects on RNA3 accumulation, RNA4synthesis and plant infection.” Unpublished Direct Submission. Submitted(19-JUN-1992) David Baulcombe, The Sainsbury Laboratory, NorwichResearch Park, Colney Lane, Norwich, NR2 7UH, United Kingdom; Hayakawaet al., Gene, 71:107 (1988); Hayakawa et al., J. Gen. Virol. 70:499(1989); Owen et al., J. Gen. Virol., 71:2243 (1990); Pappu et al., “Thenucleotide and the deduced amino acid sequences of coat protein genes ofthree Puerto Rican isolates of cucumber mosaic virus.” Unpublished(1992). This sequence is included in the GeneBank sequence data base;Salanki et al., “Complete nucleotide sequence of RNA 3 from cucumbermosaic virus strain Trk 7.” Unpublished (1993). This sequence isincluded in the GeneBank data base; Shintaku, J. Gen. Virol. 72:2587(1991)).

[0089] (c) CMV- V34

[0090] CMV-V34 RNA was prepared as described above. Subsequently, thefirst cDNA strand was synthesized using CMV-V34 template in a reactionthat included the following: approximately 2 μg CMV-V34 RNA, 1×bufferfor Superscript Reverse Transcriptase (supplied by BRL-GIBCO, GrandIsland, N.Y.), 2 mM dNTPs, oligomer primer RMM352 (37.5 μg/ml), 1.5microliters (μl) RNasin, and 1 μl Superscript Reverse Transcriptase(BRL-GIBCO) in a 20-μl reaction. After this reaction was allowed toproceed for 30 minutes, an aliquot of the first strand reaction was usedas a template in a PCR to amplify the CMV-V34 CP gene. The CMV-V34 CPgene PCR product was cloned into the pCRII vector included in the TACloning™ Kit supplied by Invitrogen Corp. Two clones were isolated forfurther study: TA17V34 and TA112V34. The CMV-V34 insert of clone TA17V34was sequenced with the use of a kit (Sequenase 2 purchased from USB,Cleveland, Ohio). Comparative sequence analysis of the CMV-V34 CP genewith other CMV CP genes (Cmvbaul, Cmvq3, Cmvw1, Cmvtrk7, Cmvfc, Cmvi17f,Cmvc, Cmvpr50, Cmvv27, Cmvp6, Cmvo, Cmvm, and Cmvy showed that theCMV-V34 CP gene is unique (Baulcombe, D. Mutational analysis of CMVRNA3: Effects on RNA3 accumulation, RNA4 synthesis and plant infection.Unpublished Direct Submission. Submitted (19-JUN-1992) David Baulcombe,The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich,NR2 7UH, United Kingdom; Hayakawa et al., Gene, 71:107 (1988); Hayakawaet al., J. Gen. Virol. 70:499 (1989); Owen et al., J. Gen. Virol.,71:2243 (1990); Pappu et al., (1992) “The nucleotide and the deducedamino acid sequences of coat protein genes of three Puerto Ricanisolates of cucumber mosaic virus.” Unpublished. This sequence isincluded in the GeneBank sequence data base; Salanki et al., “Completenucleotide sequence of RNA 3 from cucumber mosaic virus strain Trk 7.”Unpublished (1993) This sequence is included in the GeneBank data base;Shintaku, J. Gen. Virol. 72:2587 (1991)).

[0091] C. Engineering CMV CP Genes

[0092] (a) CMV- V27

[0093] The NcoI fragment in CMVV27TA21 that harbors CMV-V27 CP codingsequences was excised from CMVV27TA21 and inserted into the plantexpression cassette cpexpress in pUC18 to give CMVV27TA21ce42. Theresulting expression cassette was isolated as a partial HindIII fragmentand inserted into the binary vector pGA482G [The parent binary plasmidwas pGA482, constructed by An (Plant Physiol., 81:86 (1986)). Thisbinary vector contains the T-DNA border sequences from pTiT37, theselectable marker gene NOS-NPTII (which contains the plant-expressiblenopaline gene promoter fused to the bacterial NPTII gene obtained fromTn5), a multiple cloning region, and the cohesive ends of phage lambda(An, Plant Physiol., 81:86 (1986))] to yield pEPG191 and pEPG192.Subsequently, a PRV CP expression cassette was installed to obtain abinary vector that included both CMV-V27 CP and PRV CP expressioncassettes.

[0094] Alternatively, the CMV-V27 CP NcoI fragment obtained fromCMVV27TA21 was installed into pUC1318cp express (see Kay et al., NucleicAcids Research, 15:2779 (1987) for pUC1318; Slightom, Gene 100:251(1991) for cpexpress; pUC1318cpexpress is the cpexpress described inSlightom, however it is installed into the HindIII site of the modifiedpUC plasmid pUC 1318 described in detail in Kay et al.) to give theplasmid CMVV27TA21CE13 (similar to CMVV27TA21ce42). The plasmid pUC1318provided additional sites (e.g., BamHI and Xbal) with which the cassettecould be inserted into the binary vector pGA482G. Subsequently, thebacteria-derived gentamicin-(3)-N-acetyl-transferase gene (Allmansbergeret al., Mol. Gen. Genet., 198:514 (1985)) was installed into a SalI siteoutside of the T-DNA region, adjacent to the left border (B_(L))). TheBamHI fragment harboring the CMV-V27 CP expression cassette was isolatedand inserted into the BglII site of the binary plasmid pEPG205(PRV34/Z72/WMBN22) to give pEPG240 (CMVV27/PRV34/Z72/WMBN22). The BamHIfragment was also installed into the BglII site of the binary plasmidpEPG204 (PRV16/Z72/WMBN22) to yield pEPG239 (CMVV2716/PRV16/Z72/WMBN22)(Table 1). For further information on PRV CP genes, refer to Applicants'International Patent Application No. PCT/US95/07272 entitled “PapayaRingspot Virus Coat Protein Gene” filed on Jun. 7, 1995, incorporated byreference herein. For further information on ZYMV and WMV-2 CP genes,refer to Applicants' International Patent Application No. PCT/US89/03094filed on Jul. 20, 1989 entitled “Potyvirus Coat Protein Genes and PlantsTransformed Therewith”, incorporated by reference herein. TABLE 1 BinaryParental Plasmid Site CMVcp Cassette pEPG# pGA482G pGA482G HindIIICMVV27cpexpress 191 or 192 pPRBN pEPG204 (P16sZW) BglII CMVV27cpexpress239 pPRBN pEPG204 (P16sZW) BglII CMVV27cpexpress 240 pPRBN pEPG106 (ZW)HindIII CMVV27cpexpress 243 pGA482G pGA482G HindIII CMVV33ce7 198 pPRBNpEPG106 (ZW) HindIII CMVV33ce7 244 pPRBN pEPG204 (P16sZW) BglIICMVV27ce7 196 pPRBN pEPG205 (P34sZW) BglII CMVV27ce7 197 pGA482G pGA482GHindIII 17V34cpexp113 190

[0095] (b) CMV- V33

[0096] Subsequently, both HindIII and BamHI fragments were excised fromclone V33ce7; these fragments carried the complete expression cassettefor CMV-V33 CP gene. The BamHI fragment (V33 CP expression cassette) wasinserted into the BglII site of pEPG204 (PRV16/ZY72/WMBN22) to obtainpEPG196. The BamHI fragment was also inserted into the BglII site ofpEPG205 (PRV34/ZY72/WMBN22) to obtain pEPG197 (V3329/PRV34/ZY72/WMBN22).The HindIII fragment harboring the V33 CP cassette was installed intopGA482G to obtain pEPG198 (Table 1).

[0097] (c) CMV-V34

[0098] An NcoI fragment excised from clone TA17V34 was installed intothe NcoI site of pUC1318 cpexpress. A resulting plasmid that includesthe CMV-V34 coding NcoI fragment inserted in the sense orientation is17V34/cpexp113. A partial HindIII fragment from the plasmid17V34/cpexpll3 was isolated and installed into pGA482G to yield pEPG190(Table 1).

[0099] (d) Agrobacterium Strains

[0100] The binary plasmids described here, such as pPRBN (for furtherinformation on these plasmids, refer to Applicants' International PatentApplication No. PCT/US95/06261 entitled “Transgenic Plants ExpressingDNA Constructs Containing a Plurality of Genes to Impart VirusResistance” filed on June 7, 1995, incorporated by reference herein) ortheir derivatives, can be transferred into Agrobacterium strains A208,C58, LBA4404, C58Z707, A4RS, A4RS(pRi278b), Mog301 and others. StrainsA208, C58, LBA4404, and A4RS are available from ATCC, 12301 ParklawnDrive, Rockville, Md. A4RS (pRi278b) was obtained from Dr. F.Casse-Delbart, C.N.R.A., Route de Saint Cyr, F78000, Versailles, France.C58Z707 was obtained from Dr. A. G. Hepburn, University of Illinois,Urbana, Ill. Mog301 was obtained from Mogen NV, Leiden, Netherlands.

[0101] D. Transfer of CMV Coat Protein Genes to Tobacco

[0102] In order to test whether the CMV CP gene constructs describedherein confer protection against CMV challenge with homologous strains,some of the binary plasmids listed above (e.g., pEPG197, pEPG198,pEPG239, and pEPG240) have been used to insert these novel CMV CP genesinto Nicotiana tobacum. Agrobacterium-mediated transfer of the plantexpressible CMV CP genes described herein was done using the methodsdescribed in PCT published application WO 89/05859, entitled“Agrobacterium Mediated Transformation of Germinating Plant Seeds.”

[0103] Five R₁ progeny lines of Nicotiana t. transformed with the binaryplasmid pEPG239 and five R₁ progeny lines of Nicotiana t. transformedwith the binary plasmid pEPG240 have been obtained. These binaryplasmids include the CP gene of CMV-V27. The ten R₀ parental plants ofthese lines were assayed for NPTII protein expression by ELISA. Theyeach expressed NPTII protein by ELISA. Furthermore, these ten lines wereassayed for both the NPTII and CMV-V27 CP genes by PCR analysis. PCRanalysis detected both genes in all ten R₀ plants.

[0104] The binary plasmid pEPGl98 was used to obtain 11 R₀ transgenicNicotiana t. plants. By PCR analysis, the CMV-V33 CP gene was detectedin nine of the eleven R₀ plants tested.

[0105] E. Cloning and Engineering CMV-A35 PC Gene

[0106] 20-day-old zucchini squash plants in the greenhouse wereinoculated with CMV-A35; after 7-10 days infected leaves were harvested.Total RNA was isolated from these infected plants by the use ofTri-Reagent and the instructions provided with the reagent (MolecularResearch Center, Inc., Cincinnati, Ohio). Single-stranded cDNA wassynthesized using total RNA template. The reaction included 1 X firstStrand cDNA Synthesis Buffer (GIBCO-BRL), 1 mM dNTP's (Pharmacia), 2 μloligonucleotide primer RMM352 (150 μg/ml),2 μl RNasin (Promega), and 1μl RTase Superscript II (GIBCO-BRL) in a 20 μl reaction volume. TheCMV-A35 CP gene was PCR amplified with the use of CMV CP-specificprimers RMM351 and 352. The PCR included 3 μl of the cDNA synthesisreaction described above, 8 μl of each primer RMM351 and RMM352 (150μg/μl stock), 5 μl 10X reaction buffer, 4 μl dNTP's (10 mM), 1.5 μlMgCl₂ (50 mM), and 0.5 μl, Taq polymerase (BRL-GIBCO). PCR conditionswere carried out as follows: 93° 45 sec, 50° 45 sec, then 72° 180 secfor 30 cycles, then 72° for 5 min, then hold at 4°. PCR products werevisualized by agarose gel electrophoresis and subsequently cloned. PCRproduct molecules were cloned into the pCRII vector supplied with the TAcloning kit (Invitrogen Corp.) Four clones were identified andrestriction mapped, however, none were sequenced for further analysis.

[0107] Alternatively, an aliquot of the CMV-A35 PCR product was digestedwith NcoI and ligated it into the NcoI site of pUC19B2 cpexpress to givethe plasmid CMV-A35cpexp33. The CP insert of this plasmid was sequencedwith the use of the Sequenase II Kit supplied by USBiochemical (FIG. 8).Sequence analysis reveals that CMV-A35 CP sequence differs from the CPsequences of CMV C, V27, V33, V34, and WL (FIGS. 9 and 10). For example,A35 differs from other CMV-C strains at amino acid position #26 (FIG.9). Examination of the nucleotide sequence comparisons differs fromother CMV CP genes characterized (FIG. 10).

[0108] A BamHI/BIlII fragment was excised from A35cpexp33 and installedinto the unique BglII site pGA482G. The plasmid pUC19B2cpexp provides aBamHI site at the 5′ end of the cpexp cassette and a BglII site at the3′ end of the expression cassette. Upon insertion into a BglII site, theunique BglII site of the binary plasmid pGA482 is maintained forsubsequent insertions of gene cassettes. Binary plasmids that includethe CMV-A35 expression cassette are being transformed into variousAgrobacterium strains (e.g., C58Z707, Mog301, and LBA4404). TheseAgrobacterium strains are used to transform plants to impart resistanceto CMV CARNAS.

[0109] All publications, patents and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1 15 772 base pairs nucleic acid single linear cDNA NO NO CucumberMosaic Virus V-27 CDS 3..660 1 CC ATG GAC AAA TCT GAA TCA ACC AGT GCTGGT CGT AAC CGT CGG CGT 47 Met Asp Lys Ser Glu Ser Thr Ser Ala Gly ArgAsn Arg Arg Arg 1 5 10 15 CGT CCG CGT CGT GGT TCC CGC TCC GCC TCC TCCTCC TCG GAT GCT AAC 95 Arg Pro Arg Arg Gly Ser Arg Ser Ala Ser Ser SerSer Asp Ala Asn 20 25 30 TTT AGA GTC TTG TCG CAG CAG CTT TCG CGA CTT AACAAG ACG TTA GCA 143 Phe Arg Val Leu Ser Gln Gln Leu Ser Arg Leu Asn LysThr Leu Ala 35 40 45 GCT GGT CGT CCA ACT ATT AAC CAC CCA ACC TTT GTA GGGAGT GAA CGC 191 Ala Gly Arg Pro Thr Ile Asn His Pro Thr Phe Val Gly SerGlu Arg 50 55 60 TGT AAA CCT GGG TAC ACG TTC ACA TCT ATT ACC CTA AAG CCACCA AAA 239 Cys Lys Pro Gly Tyr Thr Phe Thr Ser Ile Thr Leu Lys Pro ProLys 65 70 75 ATA GAC CGT GGG TCT TAT TAC GGT AAA AGG TTG TTA TTA CCT GATTCA 287 Ile Asp Arg Gly Ser Tyr Tyr Gly Lys Arg Leu Leu Leu Pro Asp Ser80 85 90 95 GTC ACG GAA TAT GAT AAG AAG CTT GTT TCG CGC ATT CAA ATT CGAGTT 335 Val Thr Glu Tyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Val100 105 110 AAT CCT TTG CCG AAA TTT GAT TCT ACC GTG TGG GTA ACA GTC CGTAAA 383 Asn Pro Leu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys115 120 125 GTT CCT GCC TCC TCG GAC TTA TCC GTT GCC GCC ATC TCT GCT ATGTTC 431 Val Pro Ala Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe130 135 140 GCG GAC GGA GCC TCA CCG GTA CTG GTT TAT CAG TAT GCT GCA TCTGGA 479 Ala Asp Gly Ala Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly145 150 155 GTC CAA GCT AAC AAC AAA TTG TTG TAT GAT CTT TCG GCG ATG CGCGCT 527 Val Gln Ala Asn Asn Lys Leu Leu Tyr Asp Leu Ser Ala Met Arg Ala160 165 170 175 GAT ATA GGT GAC ATG AGA AAG TAC GCC GTC CTC GTG TAT TCAAAA GAC 575 Asp Ile Gly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser LysAsp 180 185 190 GAT GCG CTC GAG ACG GAC GAG CTA GTA CTT CAT GTT GAC ATCGAG CAC 623 Asp Ala Leu Glu Thr Asp Glu Leu Val Leu His Val Asp Ile GluHis 195 200 205 CAA CGT ATT CCC ACG TCT GGG ATG CTC CCA GTC TGA TTCCGTGTTCC 670 Gln Arg Ile Pro Thr Ser Gly Met Leu Pro Val * 210 215CAGAACCCTC CCTCCGATTT CTGTGGCGGG AGCTGAGTTG GCAGTTCTGC TATAAACTGT 730CTGAAGTCAC TAAACGTTTC ACGGTGAACG GGTTGTCCAT GG 772 218 amino acids aminoacid linear protein 2 Met Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg AsnArg Arg Arg Arg 1 5 10 15 Pro Arg Arg Gly Ser Arg Ser Ala Ser Ser SerSer Asp Ala Asn Phe 20 25 30 Arg Val Leu Ser Gln Gln Leu Ser Arg Leu AsnLys Thr Leu Ala Ala 35 40 45 Gly Arg Pro Thr Ile Asn His Pro Thr Phe ValGly Ser Glu Arg Cys 50 55 60 Lys Pro Gly Tyr Thr Phe Thr Ser Ile Thr LeuLys Pro Pro Lys Ile 65 70 75 80 Asp Arg Gly Ser Tyr Tyr Gly Lys Arg LeuLeu Leu Pro Asp Ser Val 85 90 95 Thr Glu Tyr Asp Lys Lys Leu Val Ser ArgIle Gln Ile Arg Val Asn 100 105 110 Pro Leu Pro Lys Phe Asp Ser Thr ValTrp Val Thr Val Arg Lys Val 115 120 125 Pro Ala Ser Ser Asp Leu Ser ValAla Ala Ile Ser Ala Met Phe Ala 130 135 140 Asp Gly Ala Ser Pro Val LeuVal Tyr Gln Tyr Ala Ala Ser Gly Val 145 150 155 160 Gln Ala Asn Asn LysLeu Leu Tyr Asp Leu Ser Ala Met Arg Ala Asp 165 170 175 Ile Gly Asp MetArg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp Asp 180 185 190 Ala Leu GluThr Asp Glu Leu Val Leu His Val Asp Ile Glu His Gln 195 200 205 Arg IlePro Thr Ser Gly Met Leu Pro Val 210 215 792 base pairs nucleic acidsingle linear cDNA NO NO CUCUMBER MOSAIC VIRUS v-33 CDS 3..660 3 CC ATGGAC AAA TCT GAA TCA ACC AGT GCT GGT CGT AAC CGT CGA CGT 47 Met Asp LysSer Glu Ser Thr Ser Ala Gly Arg Asn Arg Arg Arg 220 225 230 CGT CCG CGTCGT GGT TCC CGC TCC GCC CCC TCC TCC GCG GAT GCC AAC 95 Arg Pro Arg ArgGly Ser Arg Ser Ala Pro Ser Ser Ala Asp Ala Asn 235 240 245 250 TTT AGAGTC TTG TCG CAG CAG CTT TCG CGA CTT AAT AAG ACG TTG TCA 143 Phe Arg ValLeu Ser Gln Gln Leu Ser Arg Leu Asn Lys Thr Leu Ser 255 260 265 GCT GGTCGT CCA ACT ATT AAC CAC CCA ACC TTT GTA GGG AGT GAG CGT 191 Ala Gly ArgPro Thr Ile Asn His Pro Thr Phe Val Gly Ser Glu Arg 270 275 280 TGT AAATCT GGG TAC ACG TTC ACA TCT ATT ACC CTA AAG CCG CCG AAA 239 Cys Lys SerGly Tyr Thr Phe Thr Ser Ile Thr Leu Lys Pro Pro Lys 285 290 295 ATA GACCGT GGG TCT TAT TAT GGT AAA AGG TTG TTA TTA CCT GAT TCA 287 Ile Asp ArgGly Ser Tyr Tyr Gly Lys Arg Leu Leu Leu Pro Asp Ser 300 305 310 GTC ACAGAA TAT GAT AAG AAA CTT GTT TCG CGC ATT CAA ATT CGA GTT 335 Val Thr GluTyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Val 315 320 325 330 AATCCC TTG CCG AAA TTT GAT TCT ACC GTG TGG GTG ACA GTC CGT AAA 383 Asn ProLeu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys 335 340 345 GTTCCT GCC TCC TCG GAC TTA TCC GTT GCC GCC ATC TCT GCT ATG TTT 431 Val ProAla Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe 350 355 360 GCGGAC GGA GCC TCA CCG GTA CTG GTT TAT CAG TAC GCT GCA TCT GGA 479 Ala AspGly Ala Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly 365 370 375 GTCCAA GCT AAC AAC AAA TTG TTG TAT GAT CTT TCG GCG ATG CGC GCT 527 Val GlnAla Asn Asn Lys Leu Leu Tyr Asp Leu Ser Ala Met Arg Ala 380 385 390 GATATA GGC GAC ATG AGA AAG TAC GCC GTC CTC GTG TAT TCA AAA GAC 575 Asp IleGly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp 395 400 405 410GAT GCA CTC GAG ACG GAC GAG CTA GTA CTT CAT GTT GAC GTC GAG CAC 623 AspAla Leu Glu Thr Asp Glu Leu Val Leu His Val Asp Val Glu His 415 420 425CAA CGC ATT CCC ACG TCT GGG GTG CTC CCA GTA TAA T TCTGTGCTTT 670 Gln ArgIle Pro Thr Ser Gly Val Leu Pro Val * 430 435 CCAGAACCCT CCCTCCGATTTCTGTGGCGG GAGCTGAGTT GGCAGTTCTG CTGTAAACTG 730 TCTGAAGTCA CTAAACGTTTTACGGTGAAC GGGTTGTCCA TGGGTTTCGG TTTTTTTGTT 790 AA 792 218 amino acidsamino acid linear protein 4 Met Asp Lys Ser Glu Ser Thr Ser Ala Gly ArgAsn Arg Arg Arg Arg 1 5 10 15 Pro Arg Arg Gly Ser Arg Ser Ala Pro SerSer Ala Asp Ala Asn Phe 20 25 30 Arg Val Leu Ser Gln Gln Leu Ser Arg LeuAsn Lys Thr Leu Ser Ala 35 40 45 Gly Arg Pro Thr Ile Asn His Pro Thr PheVal Gly Ser Glu Arg Cys 50 55 60 Lys Ser Gly Tyr Thr Phe Thr Ser Ile ThrLeu Lys Pro Pro Lys Ile 65 70 75 80 Asp Arg Gly Ser Tyr Tyr Gly Lys ArgLeu Leu Leu Pro Asp Ser Val 85 90 95 Thr Glu Tyr Asp Lys Lys Leu Val SerArg Ile Gln Ile Arg Val Asn 100 105 110 Pro Leu Pro Lys Phe Asp Ser ThrVal Trp Val Thr Val Arg Lys Val 115 120 125 Pro Ala Ser Ser Asp Leu SerVal Ala Ala Ile Ser Ala Met Phe Ala 130 135 140 Asp Gly Ala Ser Pro ValLeu Val Tyr Gln Tyr Ala Ala Ser Gly Val 145 150 155 160 Gln Ala Asn AsnLys Leu Leu Tyr Asp Leu Ser Ala Met Arg Ala Asp 165 170 175 Ile Gly AspMet Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp Asp 180 185 190 Ala LeuGlu Thr Asp Glu Leu Val Leu His Val Asp Val Glu His Gln 195 200 205 ArgIle Pro Thr Ser Gly Val Leu Pro Val 210 215 771 base pairs nucleic acidsingle linear cDNA NO Cucumber mosaic virus V-34 CDS 3..660/codon_start= 3 /function=“ENCAPSIDATES VIRUS RNA” /product=“COATPROTEIN” /gene= “CP” /number= 1 /standard_name= “COAT PROTEIN” 5 CC ATGGAC AAA TCT GAA TCA ACC AGT GCT GGT CGT AAC CGT CGA CGT 47 Met Asp LysSer Glu Ser Thr Ser Ala Gly Arg Asn Arg Arg Arg 220 225 230 CGT CCG CGTCGT GGT TCC CGC TCC GCT TCC TCC TCT TCG GAT GCT AAC 95 Arg Pro Arg ArgGly Ser Arg Ser Ala Ser Ser Ser Ser Asp Ala Asn 235 240 245 250 TTT AGAGTC TTG TCG CAG CAG CTT TCG CGA CTT AAC AAG ACG TTA GCA 143 Phe Arg ValLeu Ser Gln Gln Leu Ser Arg Leu Asn Lys Thr Leu Ala 255 260 265 GCT GGTCGT CCA ACT ATT AAC CAC CCA ACC TTT GTA GGG AGT GAA CGC 191 Ala Gly ArgPro Thr Ile Asn His Pro Thr Phe Val Gly Ser Glu Arg 270 275 280 TGT AGACCT GGG TAC ACG TTC ACA TCT ATT ACC CTA AAG CCA CCA AAA 239 Cys Arg ProGly Tyr Thr Phe Thr Ser Ile Thr Leu Lys Pro Pro Lys 285 290 295 ATA GACCGC GGG TCT TAC TAC GGT AAA AGG TTG TTA CTA CCT GAT TCA 287 Ile Asp ArgGly Ser Tyr Tyr Gly Lys Arg Leu Leu Leu Pro Asp Ser 300 305 310 GTC ACGGAA TAT GAT AAG AAG CTT GTT TCG CGC ATT CAA ATT CGA GTT 335 Val Thr GluTyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Val 315 320 325 330 AATCCT TTG CCG AAA TTT GAT TCT ACC GTG TGG GTG ACA GTT CGT AAA 383 Asn ProLeu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys 335 340 345 GTTCCT GCC TCC TCG GAC TTA TCC GTT GCC GCC ATC TCT GCT ATG TTC 431 Val ProAla Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe 350 355 360 GCGGAC GGA GCC TCA CCG GTA CTG GTT TAT CAG TAT GCT GCA TCT GGA 479 Ala AspGly Ala Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly 365 370 375 GTTCAA GCT AAC AAC AAA TTG TTG TAT GAT CTT TCG GCG ATG CGC GCT 527 Val GlnAla Asn Asn Lys Leu Leu Tyr Asp Leu Ser Ala Met Arg Ala 380 385 390 GATATA GGT GAC ATG AGA AAG TAC GCC GTC CTC GTG TAT TCA AAA GAC 575 Asp IleGly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp 395 400 405 410GAT GCA CTC GAG ACG GAC GAG CTA GTA CTT CAT GTT GAC ATC GAG CAC 623 AspAla Leu Glu Thr Asp Glu Leu Val Leu His Val Asp Ile Glu His 415 420 425CAA CGC ATT CCC ACG TCT GGG GTG CTC CCA GTT TGA T TCCGTGTTCC 670 Gln ArgIle Pro Thr Ser Gly Val Leu Pro Val * 430 435 AGAACCCTCC CTCCGATTTCTGTGGCGGGA GCTGAGTTGG CAGTTCTGCT ATAAACTGTC 730 TGAAGTCACT AAACGTTTTACGGTGAACGG GTTGTCCATG G 771 218 amino acids amino acid linear protein 6Met Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg Asn Arg Arg Arg Arg 1 5 1015 Pro Arg Arg Gly Ser Arg Ser Ala Ser Ser Ser Ser Asp Ala Asn Phe 20 2530 Arg Val Leu Ser Gln Gln Leu Ser Arg Leu Asn Lys Thr Leu Ala Ala 35 4045 Gly Arg Pro Thr Ile Asn His Pro Thr Phe Val Gly Ser Glu Arg Cys 50 5560 Arg Pro Gly Tyr Thr Phe Thr Ser Ile Thr Leu Lys Pro Pro Lys Ile 65 7075 80 Asp Arg Gly Ser Tyr Tyr Gly Lys Arg Leu Leu Leu Pro Asp Ser Val 8590 95 Thr Glu Tyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Val Asn100 105 110 Pro Leu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg LysVal 115 120 125 Pro Ala Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala MetPhe Ala 130 135 140 Asp Gly Ala Ser Pro Val Leu Val Tyr Gln Tyr Ala AlaSer Gly Val 145 150 155 160 Gln Ala Asn Asn Lys Leu Leu Tyr Asp Leu SerAla Met Arg Ala Asp 165 170 175 Ile Gly Asp Met Arg Lys Tyr Ala Val LeuVal Tyr Ser Lys Asp Asp 180 185 190 Ala Leu Glu Thr Asp Glu Leu Val LeuHis Val Asp Ile Glu His Gln 195 200 205 Arg Ile Pro Thr Ser Gly Val LeuPro Val 210 215 25 base pairs nucleic acid single linear other nucleicacid /desc = “Oligonucleotide Primer RMM NO NO 7 CGTAGAATTC AGTCGAGCCATGGAC 25 28 base pairs nucleic acid single linear other nucleic acid/desc = ”Oligonucleotide Primer NO NO 8 GACCACTCGA GCCGTAAGCT CCATGGAC28 960 base pairs nucleic acid single linear cDNA NO NO CUCUMBER MOSAICVIRUS STRAIN C CDS 1..658 9 ATG GAC AAA TCT GAA TCA ACC AGT GCT GGT CGTAAC CAT CGA CGT CGT 48 Met Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg AsnHis Arg Arg Arg 220 225 230 235 CCG CGT CGT GGT TCC CGC TCC GCC CCC TCCTCC GCG GAT GCT AAC TTT 96 Pro Arg Arg Gly Ser Arg Ser Ala Pro Ser SerAla Asp Ala Asn Phe 240 245 250 AGA GTC TTG TCG CAG CAG CTT TCG CGA CTTAAT AAG ACG TTA GCA GCT 144 Arg Val Leu Ser Gln Gln Leu Ser Arg Leu AsnLys Thr Leu Ala Ala 255 260 265 GGT CGT CCA ACT ATT AAC CAC CCA ACC TTTGTA GGG AGT GAA CGC TGT 192 Gly Arg Pro Thr Ile Asn His Pro Thr Phe ValGly Ser Glu Arg Cys 270 275 280 AGA CCT GGG TAC ACG TTC ACA TCT ATT ACCCTA AAG CCA CCA AAA ATA 240 Arg Pro Gly Tyr Thr Phe Thr Ser Ile Thr LeuLys Pro Pro Lys Ile 285 290 295 GAC CGT GAG TCT TAT TAC GGT AAA AGG TTGTTA CTA CCT GAT TCA GTC 288 Asp Arg Glu Ser Tyr Tyr Gly Lys Arg Leu LeuLeu Pro Asp Ser Val 300 305 310 315 ACG GAA TAT GAT AAG AAG CTT GTT TCGCGC ATT CAA ATT CGA GTT AAT 336 Thr Glu Tyr Asp Lys Lys Leu Val Ser ArgIle Gln Ile Arg Val Asn 320 325 330 CCT TTG CCG AAA TTT GAT TCT ACC GTGTGG GTG ACA GTC CGT AAA GTT 384 Pro Leu Pro Lys Phe Asp Ser Thr Val TrpVal Thr Val Arg Lys Val 335 340 345 CCT GCC TCC TCG GAC TTA TCC GTT GCCGCC ATC TCT GCT ATG TTC GCG 432 Pro Ala Ser Ser Asp Leu Ser Val Ala AlaIle Ser Ala Met Phe Ala 350 355 360 GAC GGA GCC TCA CCG GTA CTG GTT TATCAG TAT GCC GCA TCT GGA GTC 480 Asp Gly Ala Ser Pro Val Leu Val Tyr GlnTyr Ala Ala Ser Gly Val 365 370 375 CAA GCC AAC AAC AAA CTG TTG TTT GATCTT TCG GCG ATG CGC GCT GAT 528 Gln Ala Asn Asn Lys Leu Leu Phe Asp LeuSer Ala Met Arg Ala Asp 380 385 390 395 ATA GGT GAC ATG AGA AAG TAC GCCGTC CTC GTG TAT TCA AAA GAC GAT 576 Ile Gly Asp Met Arg Lys Tyr Ala ValLeu Val Tyr Ser Lys Asp Asp 400 405 410 GCG CTC GAG ACG GAC GAG CTA GTACTT CAT GTT GAC ATC GAG CAC CAA 624 Ala Leu Glu Thr Asp Glu Leu Val LeuHis Val Asp Ile Glu His Gln 415 420 425 CGC ATT CCC ACA TCT GGA GTG CTCCCA GTC TGA T TCCGTGTTCC 668 Arg Ile Pro Thr Ser Gly Val Leu Pro Val *430 435 CAGAACCCTC CCTCCGATCT CTGTGGCGGG AGCTGAGTTG GCAGTTCTACTACAAACTGT 728 CTGGAGTCAC TAAACGTTTT ACGGTGAACG GGTTGTCCAT CCAGCTTACGGCTAAAATGG 788 TCAGTCGTGG AGAAATCCAC GCCAGCAGAT TTACAAATCT CTGAGGCGCCTTTGAAACCA 848 TCTCCTAGGT TTCTTCGGAA GGGCTTCGGT CCGTGTACCT CTAGCGCAACGTGCTAGTTT 908 CAGGGTACGG GTGCCCCCCC ACTTTCGTGG GGGCCTCCAA AAGGAGACCA AA960 218 amino acids amino acid linear protein 10 Met Asp Lys Ser Glu SerThr Ser Ala Gly Arg Asn His Arg Arg Arg 1 5 10 15 Pro Arg Arg Gly SerArg Ser Ala Pro Ser Ser Ala Asp Ala Asn Phe 20 25 30 Arg Val Leu Ser GlnGln Leu Ser Arg Leu Asn Lys Thr Leu Ala Ala 35 40 45 Gly Arg Pro Thr IleAsn His Pro Thr Phe Val Gly Ser Glu Arg Cys 50 55 60 Arg Pro Gly Tyr ThrPhe Thr Ser Ile Thr Leu Lys Pro Pro Lys Ile 65 70 75 80 Asp Arg Glu SerTyr Tyr Gly Lys Arg Leu Leu Leu Pro Asp Ser Val 85 90 95 Thr Glu Tyr AspLys Lys Leu Val Ser Arg Ile Gln Ile Arg Val Asn 100 105 110 Pro Leu ProLys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys Val 115 120 125 Pro AlaSer Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe Ala 130 135 140 AspGly Ala Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly Val 145 150 155160 Gln Ala Asn Asn Lys Leu Leu Phe Asp Leu Ser Ala Met Arg Ala Asp 165170 175 Ile Gly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp Asp180 185 190 Ala Leu Glu Thr Asp Glu Leu Val Leu His Val Asp Ile Glu HisGln 195 200 205 Arg Ile Pro Thr Ser Gly Val Leu Pro Val 210 215 983 basepairs nucleic acid single linear cDNA NO NO CUCUMBER MOSAIC VIRUS WHITELEAF CDS 1..657 H Kearney, C Gonsalves, D Slightom, J Quemada NucleotideSequences of the Coat Protein Genes and Flanking Regions of CucumberMosaic Virus Strains C and WL RNA 3 J. Gen. Virol. 70 1065-1073 1989 11ATG GAC AAA TCT GGA TCT CCC AAT GCT AGT AGA ACC TCC CGG CGT CGT 48 MetAsp Lys Ser Gly Ser Pro Asn Ala Ser Arg Thr Ser Arg Arg Arg 220 225 230235 CGC CCG CGT AGA GGT TCT CGG TCC GCT TCT GGT GCG GAT GCA GGG TTG 96Arg Pro Arg Arg Gly Ser Arg Ser Ala Ser Gly Ala Asp Ala Gly Leu 240 245250 CGT GCT TTG ACT CAG CAG ATG CTG AAA CTC AAT AGA ACC CTC GCC ATT 144Arg Ala Leu Thr Gln Gln Met Leu Lys Leu Asn Arg Thr Leu Ala Ile 255 260265 GGT CGT CCC ACT CTT AAC CAC CCA ACC TTC GTG GGT AGT GAA AGC TGT 192Gly Arg Pro Thr Leu Asn His Pro Thr Phe Val Gly Ser Glu Ser Cys 270 275280 AAA CCC GGT TAC ACT TTC ACA TCT ATT ACC CTG AAA CCG CCT GAA ATT 240Lys Pro Gly Tyr Thr Phe Thr Ser Ile Thr Leu Lys Pro Pro Glu Ile 285 290295 GAG AAA GGT TCA TAT TTT GGT AGA AGG TTG TCT TTG CCA GAT TCA GTC 288Glu Lys Gly Ser Tyr Phe Gly Arg Arg Leu Ser Leu Pro Asp Ser Val 300 305310 315 ACG GAC TAT GAT AAG AAG CTT GTT TCG CGC ATT CAA ATC AGG GTT AAT336 Thr Asp Tyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Val Asn 320325 330 CCT TTG CCG AAA TTT GAT TCT ACC GTG TGG GTT ACA GTT CGG AAA GTA384 Pro Leu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys Val 335340 345 CCT TCA TCA TCC GAT CTT TCC GTC GCC GCC ATC TCT GCT ATG TTT GGC432 Pro Ser Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe Gly 350355 360 GAT GGT AAT TCA CCG GTT TTG GTT TAT CAG TAT GCT GCG TCC GGA GTT480 Asp Gly Asn Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly Val 365370 375 CAG GCC AAC AAT AAG TTA CTT TAT GAC CTG TCC GAG ATG CGT GCT GAT528 Gln Ala Asn Asn Lys Leu Leu Tyr Asp Leu Ser Glu Met Arg Ala Asp 380385 390 395 ATC GGC GAC ATG CGT AAG TAC GCC GTC CTG GTT TAC TCG AAA GACGAT 576 Ile Gly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp Asp400 405 410 AAA CTA GAG AAG GAC GAG ATT GCA CTT CAT GTC GAC GTC GAG CATCAA 624 Lys Leu Glu Lys Asp Glu Ile Ala Leu His Val Asp Val Glu His Gln415 420 425 CGA ATT CCT ATC TCA CGG ATG CTC CCG ACT TAG TCCGTGTGTTTACCGGCGTC 677 Arg Ile Pro Ile Ser Arg Met Leu Pro Thr * 430 435CGAGAACGTT AAACTACACT CTCAATCGCG AGTGCTGACT TGGTAGTATT GCTTCAAACT 737GCCTGAAGTC CCTAAACGTG TTGTTGCGCG GGGAACGGGT GTCCATCCAG CTTACGGCTA 797AAATGGTCGT GTCTTTCACA CGCCGATGTC TTACAAGATG TCGAGATACC CTTGAAATCA 857TCTCCTAGAT TTCTTCGGAA GGGCTTCGTG AGAAGCTCGT GCACGGTAAT ACACTTGATA 917TTACCAAGAG TGCGGGTATC GCCTGTGGTT TTCCACAGGT TCTCCAGGTT CTCCATAAGG 977AGACCA 983 218 amino acids amino acid linear protein 12 Met Asp Lys SerGly Ser Pro Asn Ala Ser Arg Thr Ser Arg Arg Arg 1 5 10 15 Arg Pro ArgArg Gly Ser Arg Ser Ala Ser Gly Ala Asp Ala Gly Leu 20 25 30 Arg Ala LeuThr Gln Gln Met Leu Lys Leu Asn Arg Thr Leu Ala Ile 35 40 45 Gly Arg ProThr Leu Asn His Pro Thr Phe Val Gly Ser Glu Ser Cys 50 55 60 Lys Pro GlyTyr Thr Phe Thr Ser Ile Thr Leu Lys Pro Pro Glu Ile 65 70 75 80 Glu LysGly Ser Tyr Phe Gly Arg Arg Leu Ser Leu Pro Asp Ser Val 85 90 95 Thr AspTyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Val Asn 100 105 110 ProLeu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys Val 115 120 125Pro Ser Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe Gly 130 135140 Asp Gly Asn Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly Val 145150 155 160 Gln Ala Asn Asn Lys Leu Leu Tyr Asp Leu Ser Glu Met Arg AlaAsp 165 170 175 Ile Gly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser LysAsp Asp 180 185 190 Lys Leu Glu Lys Asp Glu Ile Ala Leu His Val Asp ValGlu His Gln 195 200 205 Arg Ile Pro Ile Ser Arg Met Leu Pro Thr 210 215218 amino acids amino acid single linear cDNA NO NO CUCUMBER MOSAICVIRUS Q3 AR Symons, RH Gould Cucumber Mosaic Virus RNA 3 Determinationof the nucleotide sequence provides the amino acid sequences of protein3a and viral coat protein Eur. J. Biochem 126 217-226 1982 13 Met AspLys Ser Gly Ser Pro Asn Ala Ser Arg Thr Ser Arg Arg Arg 1 5 10 15 ArgPro Arg Arg Gly Ser Arg Ser Ala Ser Gly Ala Asp Ala Gly Leu 20 25 30 ArgAla Leu Thr Gln Gln Met Leu Arg Leu Asn Lys Thr Leu Ala Ile 35 40 45 GlyArg Pro Thr Leu Asn His Pro Thr Phe Val Gly Ser Glu Ser Cys 50 55 60 LysPro Gly Tyr Thr Phe Thr Ser Ile Thr Leu Lys Pro Pro Glu Ile 65 70 75 80Glu Lys Gly Ser Tyr Phe Gly Arg Arg Leu Ser Leu Pro Asp Ser Val 85 90 95Thr Asp Tyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Ile Asn 100 105110 Pro Leu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys Val 115120 125 Pro Ser Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe Gly130 135 140 Asp Gly Asn Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser GlyVal 145 150 155 160 Gln Ala Asn Asn Lys Leu Leu Tyr Asp Leu Ser Glu MetArg Ala Asp 165 170 175 Ile Gly Asp Met Arg Lys Tyr Ala Val Leu Val TyrSer Lys Asp Asp 180 185 190 Lys Leu Glu Lys Asp Glu Ile Val Leu His ValAsp Val Glu His Gln 195 200 205 Arg Ile Pro Ile Ser Arg Met Leu Pro Thr210 215 772 base pairs nucleic acid single linear cDNA NO NO CucumberMosaic Virus A35 CDS 3..660 14 CC ATG GAC AAA TCT GAA TCA ACC AGT GCTGGT CGT AAC CGT CGA CGT 47 Met Asp Lys Ser Glu Ser Thr Ser Ala Gly ArgAsn Arg Arg Arg 220 225 230 CGT CCG CGT CGT GGT TCC CGC TCC GCC CTC TCCTCC GCG GAT GCT AAC 95 Arg Pro Arg Arg Gly Ser Arg Ser Ala Leu Ser SerAla Asp Ala Asn 235 240 245 250 TTT AGA GTC CTG TCG CAG CAG CTT TCG CGACTT AAT AAG ACG TTA GCA 143 Phe Arg Val Leu Ser Gln Gln Leu Ser Arg LeuAsn Lys Thr Leu Ala 255 260 265 GCT GGT CGT CCA ACT ATT AAC CAC CCA ACCTTT GTA GGG AGT GAA CGC 191 Ala Gly Arg Pro Thr Ile Asn His Pro Thr PheVal Gly Ser Glu Arg 270 275 280 TGT AGA CCT GGG TAC ACG TTC ACA TCT ATTACC CTA AAG CCA CCA AAA 239 Cys Arg Pro Gly Tyr Thr Phe Thr Ser Ile ThrLeu Lys Pro Pro Lys 285 290 295 ATA GAC CGT GGG TCT TAT TAC GGT AAA AGGTTG TTA CTA CCT GAT TCA 287 Ile Asp Arg Gly Ser Tyr Tyr Gly Lys Arg LeuLeu Leu Pro Asp Ser 300 305 310 GTC ACA GAA TAT GAT AAG AAG CTT GTT TCGCGC ATT CAA ATT CGA GTT 335 Val Thr Glu Tyr Asp Lys Lys Leu Val Ser ArgIle Gln Ile Arg Val 315 320 325 330 AAT CCT TTG CCG AAA TTT GAT TCT ACCGTG TGG GTG ACA GTC CGT AAA 383 Asn Pro Leu Pro Lys Phe Asp Ser Thr ValTrp Val Thr Val Arg Lys 335 340 345 GTT CCT GCC TCC TCG GAC TTA TCC GTTGCC GCC ATC TCT GCT ATG TTC 431 Val Pro Ala Ser Ser Asp Leu Ser Val AlaAla Ile Ser Ala Met Phe 350 355 360 GCG GAC GGA GCC TCA CCG GTA CTG GTTTAT CAG TAT GCC GCA TCT GGA 479 Ala Asp Gly Ala Ser Pro Val Leu Val TyrGln Tyr Ala Ala Ser Gly 365 370 375 GTC CAA GCC AAC AAC AAA CTG TTG TATGAT CTT TCG GCG ATG CGC GCT 527 Val Gln Ala Asn Asn Lys Leu Leu Tyr AspLeu Ser Ala Met Arg Ala 380 385 390 GAT ATA GGT GAC ATG AGA AAG TAC GCCGTC CTC GTG TAT TCA AAA GAC 575 Asp Ile Gly Asp Met Arg Lys Tyr Ala ValLeu Val Tyr Ser Lys Asp 395 400 405 410 GAT GCG CTC GAG ACG GAC GAG CTAGTA CTT CAT GTT GAC ATC GAG CAC 623 Asp Ala Leu Glu Thr Asp Glu Leu ValLeu His Val Asp Ile Glu His 415 420 425 CAA CGC ATT CCC ACG TCT GGA GTGCTC CCA GTC TGA T TCTGTGTTCC 670 Gln Arg Ile Pro Thr Ser Gly Val Leu ProVal * 430 435 CAGAACCCTC CCTCCGATCT CTGTGGCGGG AGCTGAGTTG GCAGTTCTGCTGTAAACTGT 730 CTGAAGTCAC TAAACGTTTT ACGGTGAACG GGTTGTCCAT GG 772 218amino acids amino acid linear protein 15 Met Asp Lys Ser Glu Ser Thr SerAla Gly Arg Asn Arg Arg Arg Arg 1 5 10 15 Pro Arg Arg Gly Ser Arg SerAla Leu Ser Ser Ala Asp Ala Asn Phe 20 25 30 Arg Val Leu Ser Gln Gln LeuSer Arg Leu Asn Lys Thr Leu Ala Ala 35 40 45 Gly Arg Pro Thr Ile Asn HisPro Thr Phe Val Gly Ser Glu Arg Cys 50 55 60 Arg Pro Gly Tyr Thr Phe ThrSer Ile Thr Leu Lys Pro Pro Lys Ile 65 70 75 80 Asp Arg Gly Ser Tyr TyrGly Lys Arg Leu Leu Leu Pro Asp Ser Val 85 90 95 Thr Glu Tyr Asp Lys LysLeu Val Ser Arg Ile Gln Ile Arg Val Asn 100 105 110 Pro Leu Pro Lys PheAsp Ser Thr Val Trp Val Thr Val Arg Lys Val 115 120 125 Pro Ala Ser SerAsp Leu Ser Val Ala Ala Ile Ser Ala Met Phe Ala 130 135 140 Asp Gly AlaSer Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly Val 145 150 155 160 GlnAla Asn Asn Lys Leu Leu Tyr Asp Leu Ser Ala Met Arg Ala Asp 165 170 175Ile Gly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp Asp 180 185190 Ala Leu Glu Thr Asp Glu Leu Val Leu His Val Asp Ile Glu His Gln 195200 205 Arg Ile Pro Thr Ser Gly Val Leu Pro Val 210 215

What is claimed is:
 1. In isolated and purified DNA molecule consistingessentially of DNA encoding the CP of the V27 strain of CMV.
 2. Theisolated and purified DNA molecule of claim 1 wherein the DNA moleculehas the nucleotide sequence shown in FIG.
 1. 3. A vector comprising achimeric expression cassette comprising the DNA molecule of claim 1, apromoter and a polyadenylation signal, wherein the promoter is operablylinked to the DNA molecule, and the DNA molecule is operably linked tothe polyadenylation signal.
 4. The vector of claim 3 wherein thepromoter is the CaMV-35S promoter.
 5. The vector of claim 4 wherein thepolyadenylation signal is the polyadenylation signal of the CaMV-35Sgene.
 6. A bacterial cell comprising the vector of claim
 3. 7. Thebacterial cell of claim 6 wherein the bacterial cell is selected fromthe group consisting of an Agrobacterium tumefaciens cell and anAgrobacterium rhizogenes cell.
 8. A transformed plant cell transformedwith the vector of claim
 3. 9. The transformed plant cell of claim 8wherein the promoter is CaMV-35S promoter and the polyadenylation signalis the polyadenylation signal of the CaMV-35S gene.
 10. A plant selectedfrom the family Cucurbitaceae comprising a plurality of the transformedcells of claim
 8. 11. A plant selected from the family Solanaceaecomprising a plurality of the transformed cells of claim
 8. 12. Anisolated and purified DNA molecule consisting essentially of DNAencoding the CP of the V33 strain of CMV.
 13. The isolated and purifiedDNA molecule of claim 12 wherein the DNA molecule has the nucleotidesequence shown in FIG.
 2. 14. A vector comprising a chimeric expressioncassette comprising the DNA molecule of claim 12, a promoter and apolyadenylation signal, wherein the promoter is operably linked to theDNA molecule, and the DNA molecule is operably linked to thepolyadenylation signal.
 15. The vector of claim 14 wherein the promoteris the CaMV-35S promoter.
 16. The vector of claim 15 wherein thepolyadenylation signal is the polyadenylation signal of the CaMV-35Sgene.
 17. A bacterial cell comprising the vector of claim
 14. 18. Thebacterial cell of claim 17 wherein the bacterial cell is selected fromthe group consisting of an Agrobacterium tumefaciens cell and anAgrobacterium rhizogenes cell.
 19. A transformed plant cell transformedwith the vector of claim
 14. 20. The transformed plant cell of claim 19wherein the promoter is CaMV-35S promoter and the polyadenylation signalis the polyadenylation signal of the CaMV-35S gene.
 21. A plant selectedfrom the family Cucurbitaceae comprising a plurality of the transformedcells of claim
 19. 22. A plant selected from the family Solanaceaecomprising a plurality of the transformed cells of claim
 19. 23. Anisolated and purified DNA molecule consisting essentially of DNAencoding the CP of the V34 strain of CMV.
 24. The isolated and purifiedDNA molecule of claim 23 wherein the DNA molecule has the nucleotidesequence shown in FIG.
 3. 25. A vector comprising a chimeric expressioncassette comprising the DNA molecule of claim 24, a promoter and apolyadenylation signal, wherein the promoter is operably linked to theDNA molecule, and the DNA molecule is operably linked to thepolyadenylation signal.
 26. The vector of claim 25 wherein the promoteris CaMV-35S promoter.
 27. The vector of claim 26 wherein thepolyadenylation signal is the polyadenylation signal of the CaMV-35Sgene.
 28. A bacterial cell comprising the vector of claim
 23. 29. Thebacterial cell of claim 28 wherein said bacterial cell is selected fromthe group consisting of an Agrobacterium tumefaciens cell and anAgrobacterium rhizogenes cell.
 30. A transformed plant cell transformedwith the vector of claim
 25. 31. The transformed plant cell of claim 30wherein the promoter is CaMV-35S promoter and the polyadenylation signalis the polyadenylation signal of the CaMV-35S gene.
 32. A plant selectedfrom the family Cucurbitaceae comprising a plurality of the transformedcells of claim
 30. 33. A plant selected from the family Solanaceaecomprising a plurality of the transformed cells of claim
 30. 34. Amethod of preparing a cucumber mosaic viral resistant plant comprising:(a) transforming plant cells with a chimeric expression cassettecomprising a promoter functional in plant cells operably linked to a DNAmolecule that encodes a CP; wherein the DNA molecule is derived from aCMV strain selected from the group consisting of V27, V33, and V34; (b)regenerating the plant cells to provide a differentiated plant; and (c)identifying a transformed plant that expresses the CMV CP at a levelsufficient to render the plant resistant to infection by the CMV strain.35. The method of claim 34 wherein the plant is a dicot.
 36. The methodof claim 35 wherein the dicot is selected from the family Cucurbitaceae.37. The method of claim 35 wherein the dicot is selected from the familySolanaceae.
 38. A vector comprising a chimeric expression cassettecomprising the DNA molecule of claim 1 and at least one chimericexpression cassette comprising a heterologous CMV CP gene, a PRV CPgene, a ZYMV CP gene, or a WMV-2 gene, wherein each expression cassettecomprises a promoter and a polyadenylation signal, wherein the promoteris operably linked to the DNA molecule, and the DNA molecule is operablylinked to the polyadenylation signal.
 39. A bacterial cell comprisingthe vector of claim
 38. 40. A transformed plant cell transformed withthe vector of claim
 38. 41. The transformed plant cell of claim 40wherein the promoter is CaMV-35S promoter and the polyadenylation signalis the polyadenylation signal of the CaMV-35S gene.
 42. A vectorcomprising a chimeric expression cassette comprising the DNA molecule ofclaim 12 and at least one chimeric expression cassette comprising aheterologous CMV CP gene, a PRV CP gene, a ZYMV CP gene, or a WMV-2gene, wherein each expression cassette comprises a promoter and apolyadenylation signal, wherein the promoter is operably linked to theDNA molecule, and the DNA molecule is operably linked to thepolyadenylation signal.
 43. A bacterial cell comprising the vector ofclaim
 42. 44. A transformed plant cell transformed with the vector ofclaim
 42. 45. The transformed plant cell of claim 44 wherein thepromoter is CaMV-35S promoter and the polyadenylation signal is thepolyadenylation signal of the CaMV-35S gene.
 46. A vector comprising achimeric expression cassette comprising the DNA molecule of claim 23 andat least one chimeric expression cassette comprising a heterologous CMVCP gene, a PRV CP gene, a ZYMV virus CP gene, or a WMV-2 CP gene,wherein each expression cassette comprises a promoter and apolyadenylation signal, wherein the promoter is operably linked to theDNA molecule, and the DNA molecule is operably linked to thepolyadenylation signal.
 47. A bacterial cell comprising the vector ofclaim
 46. 48. A transformed plant cell transformed with the vector ofclaim
 46. 49. The transformed plant cell of claim 48 wherein thepromoter is CaMV-35S promoter and the polyadenylation signal is thepolyadenylation signal of the CaMV-35S gene.